Cloning of truncated fragment of protein A

Piotr

1. Isolation of plasmids from cultures inoculated on previous day.
2. Control digest with SacI and BamHI (BamHI buffer).
3. Gel electrophoresis - proper clones founded for both products of ligation.

Checking if OmpA_omega_A_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)

Checking OmpA_omega_A_alpha and OmpA_A_alpha expression

Piotr

1. Spinning
2. Suspending
3. Adding of lysis buffer
4. Boiling
5. Putting into poliacrylamide gel
6. Transfer onto nitrocellulose
7. Blocking
8. Anti-A antibody binding
9. Washing
10. Anti-rabbit antibody binding
11. Developing with BCIP and NBT

Michał L., Ewa, Marcin

1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin.
2. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.

Preparing pACYC177+OmpA_omega_deltaA construct

Michał K.

1. Digest of pACYC177+OmpA_omega_deltaA_alpha (from 25 July) with BamHI and NotI (BamHI buffer). DNA ends blunting blunting with Klenow fragment (3 hr).
2. Gel elctrophoresis and gel-out of proper band - 4300 bp.
3. Overnight ligation of isolated DNA fragment.
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.